Demyelinating diseases of the central nervous system (CNS) such as multiple sclerosis (MS) are among the most devastating and disabling disorders, leading to severe handicap and even death. Brain damage in multiple sclerosis (MS) is marked by a destruction of the insulating sheath, known as myelin, wrapped around nerves. Although substantial efforts have centered on suppression of the immune response that attacks myelin, it is becoming clear that it does not address the major problem of the disease: loss of myelin. CNS myelin is produced by specialized cells called oligodendrocytes. A common feature of demyelinated lesions is the differentiation block of oligodendrocyte precursors (OPC) at a pre-myelinating stage. Thus, the identification of the critical factors that promote oligodendrocyt production from OPCs and block the inhibitory signals for myelination will help to improve myelin repair and devise effective treatment strategies for demyelinating diseases. Ultimately the myelination program is controlled at the level of gene regulation. Histone deacetylases play an important role in the regulation of gene expression by modifying both histones and non-histone regulatory proteins, thereby controlling how cells grow and differentiate. Recently we demonstrated that class I histone deacetylases, HDAC1/2, are essential for OPC proliferation and differentiation. Our preliminary data indicate that deletion of another class I histone deacetylase, Hdac3, in the oligodendrocyte lineage resulted in severe myelination deficits. Our central hypothesis is that Hdac3 and its mediators are required for establishing oligodendrocyte identity and controlling the myelination process in the CNS. The objective of this application is to gain a crucial insight into the molecular basis of the myelination process regulated by Hdac3 and its co-factor complex. We will utilize in vivo mutagenesis approaches to define the role of Hdac3 in CNS myelination and myelin repair by analyzing new conditional knockout mouse strains, NG2-CreERT:Hdac3 flox/flox and Plp-CreERT:Hdac3 flox/flox mice, in which Hdac3 is selectively ablated in OPC and differentiating OL, respectively, in a developmentally-controlled manner. In addition, we will identify and characterize the direct targets of Hdac3 that control the myelination program by employing genome-wide RNA-sequencing (RNA-seq) and chromatin-immunoprecipitation-sequencing (ChIP-seq) strategies. The long-term goal of the research proposed here is to foster the development of agents that modulate the activity of Hdac3 and its downstream effectors to promote myelination and, of urgent clinical relevance, remyelination. The proposed studies will not only advance our understanding of the mechanisms of CNS myelination, but also identify potential therapeutic targets to promote oligodendrocyte regeneration and myelin repair for the patients with demyelinating diseases such as MS, leukodystrophies, stroke, and injury to the brain or spinal cord.